The apparent rate constant for the dissociation of force generating myosin crossbridges from actin decreases during Ca2+ activation of skinned muscle fibres

Summary The effect of Ca²⁺ activation on the apparent rate constant governing the dissociation of force generating myosin cross-bridges was studied in skinned rabbit adductor magnus fibres (fast-twitch) at 21±1 °C. Simultaneous measurements of Ca²⁺-activated isometric force and ATPase activity were conducted in parallel with simultaneous measurements of DANZ-labelled troponin C (TnC_DANZ) fluorescence and isometric force in fibres whose endogenous troponin C had been partially replaced with TnC_DANZ. The Ca²⁺ activation of isometric force occurred at approximately two times higher Ca²⁺ concentration than did actomyosin ATPase activity at 2.0 mM MgATP. Since increases in both TnC_DANZ fluorescence and ATPase activity occurred over approximately the same Ca²⁺ concentration range at substantially lower concentrations of Ca²⁺ than did force, this data suggests that the TnC_DANZ fluorescence is associated with the Ca²⁺ activation of myosin crossbridge turnover (ATPase) rather than force. According to the model of Huxley (1957) and assuming the hydrolysis of one molecule of ATP per cycle of the crossbridge, the apparent rate constantg_app for the dissociation of force generating myosin crossbridges is proportional to the actomyosin ATPase/isometric force ratio. This measure ofg_app shows approximately a fivefold decrease during Ca²⁺ activation of isometric force. This change ing_app is responsible for separation of the Ca²⁺ sensitivity of the normalized ATPase activity and isometric force curves. If the MgATP concentration is reduced to 0.5 mM, the change ing_app is reduced and consequently the difference in Ca²⁺ sensitivity between normalized steady state ATPase and force is also reduced.The abbreviations used are TnC troponin C - DANZ 5-dimethylaminonapthalene-2-sulphonyl aziridine - TnC_DANZ DANZ-labelled TnC - a the number of half sarcomeres - A the cross-sectional area of the fibre - AP₅A P¹,P⁵-di(adenosine-5)pentaphosphate - EGTA ethyleneglycolbis-(betaaminoethyl ether)-N,N,N,N-tetraacetic acid - F force a muscle fibre develops - f_app apparent rate of formation of force generating myosin crossbridges - F_av the average force per myosin head - Fs steady-state fraction of cycling myosin crossbridges in the force generating state - g_app apparent rate of dissociation of force generating myosin crossbridges - L_1/2s the length of a half sarcomere - LDH lactate dehydrogenase - [M] is the concentration of myosin per fibre volume - NAD nicotinamide adenine dinucleotide - NADH reduced form of NAD - pCa -log₁₀ of the free Ca²⁺ concentration - PEP phosphenol pyruvate - PK pyruvate kinase